Quantification of Biotin in Feces

Biotin content in feces was measured by a bioassay using Lactobacillus plantarum ATCC 8014. Biotin assay medium “Nissui” (Nissui pharmaceutical, Tokyo, Japan) was used according to the manufacturer’s instructions. Free form biotin in feces was extracted with water at 60°C for 10 min and the fecal suspension was centrifuged and filtered using a 0.45 μm pore-size filter. Some biotin exists in samples in the protein-bound form, and therefore to measure total biotin the feces was treated with 4.5N H₂SO₄ at 121°C for 1 h using an autoclave then neutralized with the same volume of 4.5 N NaOH to release bound biotin then assayed as above.